Owing to the advances in fundamental research into drug innovation, including molecular biology and genomics (genome science), the circumstances of drug innovation have changed dramatically over these several years, and new approaches to drug innovation, represented by genome drug innovation, are being developed.
However, it remains impossible to predict protein functions (physiological actions) from the nucleotide sequences clarified by genomics; there is a post-genome need for the establishment of techniques that link genetic information to new drugs. As such, proteomics (protein analysis science) is drawing attention, which aims at isolating and identifying all proteins (reportedly more than 100,000 kinds) translated from the aforementioned nucleotide sequences, and assigning their functions.
Although proteome means all proteins that constitute a single organism in a narrow sense, it sometimes means, in a broad sense, all proteins contained in a histologically or anatomically specified portion of an organism, like all proteins in the cell fluid of a particular cell, all proteins contained in serum, all proteins contained in a particular tissue, and the like. In the present specification, this term is used in the broad sense, but it is evident that the complete assembly of proteomes in the broad sense is the proteome in the narrow sense.
In proteome analysis, a method combining two-dimensional electrophoresis and mass spectrometry has conventionally been used commonly, but the following problems remain unresolved. That is, in the conventional method, the migration gel must be divided into fractions, from each of which proteins must be extracted with a special solution, before the sample is subjected to mass spectrometry after migration. Due to the long time taken and the requirement of painstaking operation in multiple stages, measuring time shortening, apparatus size reduction, screening of a large number of analytes, and whole-apparatus automation have been extremely difficult.
Furthermore, there is another problem of what is called “low-abundance proteins”. In yeast, for example, only 100 genes produce 50% of the total protein weight of the yeast. This means that the remaining 50% proteins are products of thousands of genes. A large amount of low-abundance proteins contains a large amount of proteins that are most important to the body, such as regulatory proteins and signal transmission proteins, including receptors. However, the situation with the conventional method is such that the large number and trace amount of protein samples separated by electrophoresis cannot be recovered.
In an attempt to overcome these limitations in the combined technology of electrophoresis and mass spectrometry, and to clarify protein-protein interactions, a variety of efforts are now being made. For example, the isotope-coded affinity tag method (ICAT: isotope-coded affinity tag; Nat. Biotech., 17: 994-999 (1999)), the two-hybrid system in yeast, BTA-MS-MS, the protein array method (solution, chip; Trends Biotechnol., 19: S34-39 (2001)), peptidomics by LC-MS-MS, and the like are available. In particular, as examples of the protein array method, the solid phase protein array method (chip method; Curr. Opin. Biotechnol., 12: 65-69 (2001)), the liquid phase array method (fluorescence-encoded beads; Clin. Chem., 43: 1749-1756 (1997); Nat. Biotech., 19: 631-635 (2001); barcoded nanoparticles; Trends Biotechnol. (2001), ibidem), wherein information has been incorporated in nano-particles, and the like can be mentioned.
Meantime, the present inventors have proposed a method of proteome analysis that enables simultaneous analysis of membrane proteins and compounds capable of interacting therewith by grouping them (WO 02/56026).
Aside from such improvements in measuring methods (systems), there are some reports of attempts for improvements in the field of protein mass spectrometry, though they are not intended for proteome analysis, or are technically difficult to apply to proteome analysis. Methods have been reported, which comprise blotting proteins separated by electrophoresis to a polyvinylidene difluoride (PVDF) membrane, and taking measurements with the membrane immobilized to a stainless steel plate for MALDI type mass spectrometry using a double-coated tape (Electrophoresis, 17: 954-961 (1996)), a frame (Anal. Chem., 69: 2888-2892 (1997)) or grease (Anal. Chem., 71: 4800-4807 (1999); Anal. Chem., 71: 4981-4988 (1999); WO 00/45168). These methods are faulty in that not only the procedures are painstaking due to immobilization of the PVDF membrane to the plate for mass spectrometry in the midst of measurement, but also the background is high and the relative peak intensity is extremely low, so that they are unsuitable to proteome analysis, which requires high detection sensitivity.
Also, a method comprising blotting a gel after electrophoresis directly to a matrix-coated plate for mass spectrometry and conducting mass spectrometry, and a method comprising electrically blotting a protein to a PVDF membrane for blotting, and then diffusion-blotting the protein to a matrix-coated plate for mass spectrometry, have been reported (U.S. Pat. No. 5,595,636). However, in these methods, when electrical blotting is used, there is concern that the matrix dissolves in the blotting buffer during blotting and measurements themselves become impossible. On the other hand, when diffusion blotting is used, the blotting efficiency is low so that the detection sensitivity is insufficient particularly to blotting low-abundance proteins to a plate for mass spectrometry.
Furthermore, a method based on an improvement of the above-described method has been reported, which comprises applying a mixture of nitrocellulose (a membrane component for blotting use) and a matrix to a plate for mass spectrometry as (GB 2312782 A). However, even this method cannot be said to have completely resolved the problems, in the case of electrical blotting or diffusion blotting.
As plates for mass spectrometry, commonly used aluminum or stainless steel plates, as well as improvements thereof, such as plates for mass spectrometry coated with silica, or having a hydrophobic group added thereto, are commercially available (manufactured by Ciphergen, WO 94/28418). However, even these technologies and products do not always meet the research and development needs for conducting proteome analysis in large amounts, with quickness, and at high sensitivity.
An object of the present invention is to introduce a new technology to the conventional method of proteome analysis based on a combination of electrophoresis and mass spectrometry. Specifically, the object of the present invention is to provide a technology that enables the conduct of proteome analysis in large amounts, with quickness, and at high sensitivity.